SK287689B6 - Immunostimulatory oligodeoxynucleotides - Google Patents

Abstract

An use of oligodeoxynucleic acid molecule (ODN) having the structure according to formula (I), wherein any X is O or S; any NMP is a 2' deoxynucleoside monophosphate or monothiophosphate, selected from the group consisting of deoxyadenosine-, deoxyguanosine-, deoxyinosine-, deoxycytosine-, deoxyuridine-, deoxythymidine-, 2-methyl-deoxyinosine-, 5-methyl-deoxycytosine-, deoxypseudouridine-, deoxyribosepurine-, 2-amino-deoxyribosepurine-, -6-S-deoxyguanine-, 2-dimethyl- deoxyguanosine- or N- isopentenyl-deoxyadenosine-monophosphate or -monothiophosphate, NUC is a 2' deoxynucleoside, selected from the group consisting of deoxyadenosine-, deoxyguanosine-, deoxyinosine-, deoxycytosine-, deoxyuridine-, deoxythymidine-, 2-methyl-deoxyinosine-, 5-methyl-deoxycytosine-, deoxypseudouridine-, deoxyribosepurine-, 2-amino-deoxyribosepurine-, 6-S-deoxyguanine-, 2-dimethyl- deoxyguanosine- or N-isopentenyl-deoxyadenosine; a and b are integers from 0 to 100 with the proviso that a + b is between 4 and 150, B and E are common groups for 5' or 3' ends of nucleic acid molecules, for preparing an immunostimulatory pharmaceutical composition. An immunostimulatory pharmaceutical composition containing such ODN.

Description

Technical field

The present invention relates to the use of an oligodeoxynucleic acid (ODN) molecule for the manufacture of an immunostimulatory pharmaceutical composition and a pharmaceutical composition comprising such ODN molecules.

BACKGROUND OF THE INVENTION

Vaccines can save more lives (and resources) than any other medical intervention (Nossal, 1998). Worldwide vaccination programs have drastically reduced the incidence of many fatal diseases. Although this is true for a variety of diseases such as tuberculosis, diphtheria, whooping cough, measles and tetanus, there are no effective vaccines for many infectious diseases involving most viral infections, such as AIDS. There are no effective vaccines for other diseases, infectious or non-infectious, requiring the lives of millions of patients each year, including malaria and cancer. Extremely fast developing bacteria and antibiotic resistant microorganisms call for alternative treatments where vaccines are the logical choice. Finally, the great need for vaccines is also illustrated by the fact that infectious diseases, rather than cardiovascular diseases or cancer, or injuries, remain the largest cause of death and disability in the world (Bloom and Widdus, 1998).

From the immunological point of view, the biggest problem today in the field of vaccines is that traditional vaccines (and / or immunomodulatory compounds contained in these preparations) are designed to induce high levels of antibodies (Harrow and Lane (1988). But the antibodies themselves are not effective. in the prevention of a large number of diseases involving most diseases caused by viruses, intracellular bacteria, certain parasites and tumors Examples of such diseases are, but are not limited to, said HIV or Plasmodium spec. in the case of malaria. In some cases, the cellular part of the immune system, including the T cells, is responsible rather than the antibody part, and therefore new innovative technologies are needed to overcome the limitations of conventional vaccines, the focus must be on technologies that reliably induce cellular immune ystem, including antigen-specific T cells, which recognize molecules on cells infected with pathogens. Vaccines are ideally designed to induce T cells recognizing diseased and / or infected cells from normal cells and at the same time antibodies secreted by B cells recognizing pathogens in the extracellular compartments.

Some established vaccines contain living attenuated organisms where there is a risk of reversal to a virulent wild strain. This may have life-threatening consequences especially for immunosuppressed hosts. Vaccines are alternatively administered as combinations of pathogen-derived antigens together with compounds that induce or enhance immune responses against these antigens (these compounds are commonly called adjuvants) because these subunit vaccines are not generally effective in themselves.

While there is no doubt that these vaccines are valuable medical therapeutics, it is a disadvantage that due to their complexity, serious side effects can be induced, for example, to antigens that are contained in a vaccine showing cross-reactivity with molecules expressed by the cells of vaccinated individuals. In addition, it is difficult to meet existing requirements of regulatory authorities such as the World Health Organization (WHO), the US Food and Drug Administration (FDA) and their European counterparts regarding the precise specification of vaccine composition and immune-induction mechanisms.

Antigen-presenting cells belong to the innate immune system that developed as the first host defense line and that limits infection soon after exposure to microorganisms (Hofmann et al., 1999). Cells of the innate immune system recognize patterns or relatively non-specific structures expressed on their targets, rather than more sophisticated, specific structures that are recognized by the adaptive immune system (Hoffmann et al., 1999). Examples of cells of the innate immune system are macrophages and dendritic cells, but also granulocytes (e.g. neutrophils), natural killer cells, and other cells. In contrast, cells of the adaptive immune system recognize specific antigenic structures including peptides in the case of T cells and peptides as well as three-dimensional structures in the case of B cells. The adaptive immune system is more specific and sophisticated than the innate immune system and improves after repeated exposure to a given pathogen / antigen. Phylogenetic is the innate immune system much older and can be found in very primitive organisms. However, the innate immune system is critical during the initial phase of antigenic exposure because, in addition to the fact that the innate immune system cells (APCs) retain pathogens, they also provide the cells of the adaptive immune system with information and thus trigger specific immune responses leading to the elimination of intruders. In summary, cells of the innate immune system, and in particular APC cells, play a critical role during the induction phase of immune responses by (a) arresting infectious agents with a primitive pattern recognition system, and

b) providing the cells of the adaptive immune system with information leading to specific immune responses and memories resulting in the release of emerging pathogens or other targets (Roitt et al., 1998). These mechanisms may also be important for the clearance or retention of tumor cells.

As mentioned, cells of the innate immune system recognize patterns expressed on their respective targets. Examples are lipopolysaccharides (LPS) for Gram negative bacteria, mycobacterial glycolipids, lipoteichoic acids Gram positive bacteria, mannan of yeast, and double-stranded RNA viruses (Hoffmann et al., 1999). In addition, they can recognize structures such as impaired protein glycosylation on tumor cells.

Recent findings describe DNA of protozoa or lower eukaryotes as other structures recognized by the innate (but possibly also adaptive) immune system of mammals (and probably by most, if not all vertebrates) (Krieg, 1966; Lipford et al., 1998).

The immune system recognizes lower organisms including bacteria, probably due to structural and sequence differences between the pathogen and the host DNA. The target is particularly short DNA segments derived from non-vertebrate animals or in the form of short synthetic ODNs containing unmethylated cytosine-guanine dinucleotides (CpG) in a particular base context (Krieg et al., 1995). CpG motifs are found at the expected frequency in bacterial DNA, but are much less common in vertebrate DNA (LipLord et al., 1998; Pisetsky, 1998). In addition, non-vertebrate CpG motifs (i.e., bacterial) are not methylated, while vertebrate CpG sequences are methylated. These differences between bacterial DNA and vertebrate DNA allow vertebrates to recognize non-vertebrate DNA as a dangerous signal.

Natural CpG-containing DNAs, ODNs as well as thiophosphate-substituted (replacement of thiophosphate residues with phosphate) ODNs containing CpG motifs (CpG-ODN) are not only potent activators of immune cell proliferation and humoral immune responses (Krieg et al., 1995), but also stimulate strong cellular immune responses (review in Lipford et al., 1998). DNA / ODN containing unmethylated CpG motifs can directly activate monocyte cells (dendritic cells, macrophages) and B cells. Natural killer cells are not likely to be directly activated, but respond to monocyte-derived IL-12 (interleukin 12) with a large increase in their IFN-γ production (Chace et al., 1997). As a result, induction of monocytes and NK cells by CpG DNA promotes the induction of Th1-type responses and the development of cytotoxic T cells.

It is known that ribosucleic acid based on inosine and cytosine, such as polyinosine-poylcytidylic acid (poly I: C), promotes Th1-specific immune responses. It is known to stimulate macrophages to produce cytokines such as IL--α and IL-12 (Manetti et al., 1995), also known as a potent inducer of type I interferon (Manetti et al., 1995) and potent NK cell stimulator (Cavanauah et al., 1996).

However, this effect was strictly limited to ribonucleic acid containing inosine and cytidine residues (WO98 / 16247).

Investigations by researchers of the present invention have shown that ODNs containing unmethylated CpG motifs, which, although effectively stimulating the immune system, have basic disadvantages, particularly with respect to specificity (high background) and induction of side effects, such as high systemic TNF-α production. It is known that high systemic release of TNF-α causes toxic shock syndrome, which can cause death of affected patients.

It is therefore an object of the present invention to provide suitable novel ODNs that do not have such drastic side effects as ODNs based on CpG sequences. It is therefore a further object to reduce the side effects of pharmaceutical preparations containing known ODNs and to provide safe and efficacious well tolerated pharmaceutical preparations with effective immunostimulatory properties that are suitable for the vaccination of animals, especially mammals, including humans.

SUMMARY OF THE INVENTION

Accordingly, the invention provides the use of an oligodeoxynucleic acid (ODN) molecule having the structure defined by formula (I)

wherein any X is O or S;

any of the NMP residues is 2'-deoxynucleoside monophosphate or -monothiophosphate selected from deoxyadenosine-, deoxyguanosine-, deoxyinosine-, deoxycytosine-, deoxyuridine-, deoxythymidine-, 2-methyl-deoxyinosine-, 5-methyl-deoxycytosine- , deoxyribosapurine, 2-aminodeoxyribosapurine, 6-S-deoxyguanine, 2-dimethyldeoxyguanosine or N-isopentenyldeoxyadenosine monophosphate or monothiophosphate,

NUC is a 2'-deoxynucleoside selected from deoxyadenosine-, deoxyguanosine-, deoxyinosine-, deoxycytosine-, deoxyuridine-, deoxythymidine-, 2-methyldeoxyinosine-, 5-methyldeoxycytosine-, deoxypseudouridine-, deoxypyrosibine-, deoxyribosine-, deoxyrurosine, 6-deoxyurosine, -deoxyguanine-, 2-dimethyldeoxyguanosine- or N-isopentenyldeoxyadenosine, and b and are integers from 0 to 100 exclusively that a + b is a number between 4 and 150,

B and E are common groups for the 5 'or 3' ends of the nucleic acid molecules to produce an immunostimulatory pharmaceutical composition.

Surprisingly, it has been shown that ODNs containing deoxyinosine residues (i-ODNs) have an immunostimulatory effect comparable, or in many cases even better, than ODNs containing CpG motifs. In addition, the ODNs of the present invention produce more specific immune responses to a given antigen or antigenic fragment than a CpG ODN. In addition, the ODNs of the present invention reduced the induction of unwanted side reactions, in particular, the induction of systemic TNF-α or IL-6.

While certain immunostimulatory effects have been described for RNA molecules containing RNA molecules, such as poly-IC or the molecules mentioned in WO98 / 16247, it has surprisingly been found that deoxynucleic acid molecules containing deoxyinosine residues can be good immunostimulatory ODNs.

In addition, the I-ODNs of the present invention are independent of the specific motif or palindromic sequence described for CpG oligonucleotides over ODNs based on specific CpG motifs (see, for example, EP 0 468 520 A2, WO96 / 02555, WO98 / 18810, WO98 / 37919, WO 98 / 40100, WO98 / 52581, WO99 / 51259 and WO99 / 56755, all incorporated herein by reference). Therefore, one group of I-ODNs of the present invention may preferably contain a Cl motif (and therefore preferred forms of the present ODNs are described in these incorporated references, where one or more guanosine residues are replaced by deoxyinosine residues). This motif is not necessary for its major immunostimulatory property since I-ODNs with inosine not located in the Cl or IC context have immunostimulatory properties as well.

The I-ODN of the present invention is therefore a DNA molecule containing a deoxyinosine residue, which is preferably provided in single-stranded form.

The I-ODN of the present invention may be isolated by recombinant methods or may be chemically synthesized, in the latter case the I-ODN of the present invention may contain modified oligonucleotides that may be synthesized using standard chemical transformations such as methylphosphonates or other modified oligonucleotides phosphorus-based compounds such as phosphotriesters, phosphoamidates and phosphorothiorates. Other modified phosphorus-free oligonucleotides may also be used (Stirchak et al., March 17 (1989), 6139-6141), but monophosphates and monothiophosphates are preferred 2 'deoxynucleoside monophosphates for use in the present invention.

The NMPs of the I-ODNs of the present invention are preferably selected from the group consisting of deoxyadenosine, deoxyguanosine, deoxyinosine, deoxycytosine, deoxyuridine, deoxythymidine, 2-methyl-deoxyinosine, 5-methyl-deoxycytosine monophosphate (or monothiophosphate). such as the 5'-deoxyribose phosphate or thiophosphate group). While for CpG motif-based ODNs the basis is that this motif is unmethylated, this is not surprisingly the case with the ODN of the present invention where, for example, 2-methyl-deoxyinosine or 5-methyl-deoxycytosine residues generally have no negative effect on the immune-stimulatory properties of ODNs. according to the present invention. Alternatively, other particularly inert groups such as -F, -NH ₂ , -CH ₃ , especially -CH _3, may also be present at the 2-position of the ribose group instead of the 2-deoxyphor NMPs. However, the -OH and SH groups are excluded for the I-ODN of the present invention from being located at the 2'-ribose position, especially the ribose residue for the inosine NMP.

The ODN length of the present invention is within the range of standard ODNs used to date. Therefore, molecules with a total length below 4 and above 150 bases have a progressively decreasing immunostimulatory potential. Preferred ODNs comprise between 10 and 60 bases, in particular, between 15 and 40 bases (nucleotides), resulting in a + b in formula (I) in these preferred forms between 10 and 60 bases, preferably between 15 and 40 bases.

While the inosine-containing and cytidine-containing ribonucleic acid molecules described in the prior art as immunostimulatory molecules are large and relatively undefined polynucleic acids with molecular weights well above 200,000 (commercially available polyinosine-polycytidylic acid from Sigma Chemicals, it has a molecular weight in between 220 000 and 460 000 (at least 500-1 000 C + I residues)). The molecules of the present invention are DNA molecules that are much shorter with a well defined length and composition, and are highly reproducible in the products.

It is further preferred that the NMP-containing deoxyinosine of the I-ODN of formula (I) is a monothiophosphate with one or four sulfur atoms, and also that other NMPs, particularly all other NMPs, are present as nucleo4 zid monothiophosphates because such ODNs have higher resistance to nucleases (it is clear for the present invention that the term "mono" in the term "monothiophosphates" refers to phosphate, that is, one phosphate group (one phosphorus atom) is present in each NMP). Preferably, at least one of X 1 and X ₂ is S and at least one X ₃ and X ₄ is O in the NMP of the present invention. X ₃ and X ₄ are preferably O. X ₃ may (due to NMP synthesis) be derived, for example, from the phosphate group or from the 3-group of the NMP ribose).

ODNs according to the present invention preferably comprise the sequence hhh wdi dhh h, nhh hhh wdi nhh hhh hhh wn, nhh wdi din hhh hdi nh nh, nhh hhh wdi dhh hhh hhh wn or nhh wdi did hhh hdi ddi dh, where any is a deoxynucleoside monophosphate or monothiophosphate selected from the group consisting of deoxyadenosine-, deoxyguanosine-, deoxyinosine-, deoxycytosine- or deoxythymidine monophosphate, or -monothiophosphate; , or monothiophosphate, i is deoxyinosine monophosphate or -monothiophosphate, any w is a 2 'deoxynucleoside monophosphate or monothiophosphate selected from the group consisting of deoxyadenosine or deoxythymidine monophosphate, or -monothiophosphate, and any d is 2' deoxynucleoside monophosphate or monophosphate containing deoxyadenosine-, deoxyguanosine- or deoxythymidine-monofo or monothiophosphate.

As mentioned, a specific motif (such as CpG or palindrome) is not required for the I-ODN of the present invention. However, ODNs containing a Cl motif are preferred, so that in a preferred form, the ODN of formula (I) comprises at least one 2 'deoxycytosine monophosphate or monothiophosphate, 3'-adjacent to 2' deoxyinosine monophosphate or monothiophosphate to form 5'-CI 3 ' - motif.

Preferred ODNs of the present invention comprise one or more of gacitt, iacitt, gaictt, iaictt, wherein a is deoxyadenosine monophosphate or -monothiophosphate, g is deoxyguanosine monophosphate or -monothiophosphate, and is deoxyinosine monophosphate or -monothiophosphate, -monophosphate or -monothiophosphate whether it is deoxythymidine monophosphate or -monothiophosphate.

The I-ODNs of the present invention are particularly suitable for application in the pharmaceutical field, for example, as an medicament to an animal or a human. They are specifically adapted to act as an immuno-stimulant, particularly in or with the vaccine formulation.

Therefore, the present invention also relates to a pharmaceutical composition comprising an ODN of the present invention.

Since the preferred pharmaceutical composition of the present invention is a vaccine, the composition should comprise an antigen instead of an ODN of the present invention. The potential of this antigen to enhance the protection / immune response of a vaccinated individual is strongly enhanced by its combination with the ODN of the present invention, particularly due to their immunostimulatory activity.

The vaccine may contain a variety of different antigens. Examples of antigens are killed by whole organisms, such as inactivated viruses or bacteria, fungi, protozoa or even tumor cells. Antigens may also consist of subfractions of these organisms / tissues from proteins, or in simpler form, from peptides. Antigens may also be recognized by the immune system in the form of glycosylated proteins or peptides and may also be or contain polysaccharides or lipids. Short peptides may be used because, for example, cytotoxic T cells (CTLs) recognize antigens in the form of short usually 8-11 amino acids long peptides in association with the major histocompatibility complex (MHC) (Rammensee et al., Immunogenetics 41, (1995), 178- 228). B cells recognize longer peptides from about 15 amino acids (Harrow et al., Cold Spring Harbor: Cold Spring Harbor Laboratory, (1988)). Unlike T cell epitopes, the three-dimensional structures of B cell antigens may also be important for antibody recognition. To obtain persistent antigen-specific immune responses, adjuvants are useful for triggering immune cascades that include all necessary cells of the immune system. Adjuvants act primarily, but are not limited to their mode of action, on so-called antigen-presenting cells (APCs). These cells usually first encounter the antigen (s) with the following presentation of the processed or unmodified antigen to the immune effect. Transient cell types can also play a role. Only effector cells of appropriate specificity are activated in a productive immune response. The adjuvant may also locally retain antigens and co-injected with other factors. In addition, the adjuvant may act as a chemoattractant for other immune cells, or may act locally and / or systematically as a stimulant for the immune system.

According to a preferred embodiment, T cell epitopes are used as antigens. Alternatively, a combination of T cell epitopes and B cells may be preferred.

The antigens to be used in the present compositions are not critical. Of course, mixtures of different antigens may also be used according to the present invention. Proteins or peptides derived from a viral or bacterial pathogen, or from fungi or parasites are preferably used as such antigens (including derivatized antigens or glycosylated, or lipid antigens, polysaccharides or lipids). Another preferred source of antigens is tumor antigens. Preferred pathogens are selected from the group consisting of human immunodeficiency virus (HIV), hepatitis A and B viruses, hepatitis C virus (HCV), Rous sarcoma virus (RSV, Epstein Barr virus (EBV), influenza virus, rotavirus, Staphylococcus aureus Chalmydia trachomatis, Mycobacterium tuberculosis, Streptococcus pneumoniae, Bacillus antracis, Vibrio cholerae, Plasmodium sp. (Pl. Falciparum, Pl. Vivax, etc./ Aspergillus sp. Or Candida albicans. Antigens may also be molecules expressed by tumor cells). The derivative process may include purification of a specific protein from pathogenic / tumor cells, pathogen inactivation as well as proteolytic or chemical derivatization or stabilization of such a protein In the same way, tumor antigens (tumor vaccines) or autoimmune antigens may also be used in the pharmaceutical composition of the present invention. Tumor vaccination or treatment of autoimmune diseases may be performed with such formulations.

For peptide antigens, the use of peptide mimitopes / agonists / superagonists / antagonists or peptides altered at certain positions without affecting the immunological properties of non-peptide mimitopes / agonists / superagonists / antagonists (reviewed in Sparbier and Walden, 1999) is included in the present invention. The peptide antigens may also comprise an extension at either the carboxy or amino terminus of the peptide antigen to facilitate interaction with the polycationic compound (s) or the immunostimulatory compound (s). Peptide-based antagonists can be used to treat autoimmune diseases.

Antigens may also be derivatized to include molecules that enhance antigen presentation and target antigens to antigen presenting cells.

In one embodiment, the pharmaceutical composition serves to gain tolerance to proteins or protein fragments and peptides that play a role in autoimmune diseases. The antigens used in these forms serve to allow immune system tolerance or reduce immune responses against epitopes playing a role in autoimmune processes.

Preferably, the pharmaceutical composition of the present invention, especially in the form of a vaccine, further comprises a polycationic polymer, preferably a polycationic peptide, in particular a polyarginine, polylysine or an antimicrobial peptide.

The polycationic compound (s) to be used according to the present invention may be any polycationic compound having the characteristic effect of WO 97/30721. Preferred polycationic compounds are selected from basic polypeptides, organic polycations, basic polyamino acids, or mixtures thereof. These polyamino acids should have a chain length of at least 4 amino acid residues (see: Tuftsin as described in Goldman et al (1983)). Particularly preferred are substances containing peptide bonds such as polylysine, polyarginine and polypeptides containing more than 20%, especially more than 50% basic amino acids in the range of more than 8, especially more than 20 amino acid residues or mixtures thereof. Other preferred polycations and their pharmaceutical compositions are described in WO 97/30721 (e.g. polyethyleneimines) and WO 99/38528. Preferably, these polypeptides comprise between 20 and 500 amino acid residues, especially between 30 and 200 residues.

These polycationic compounds may be produced chemically or recombinantly, or may be derived from natural sources.

The cationic (poly) peptides may also be polycationic antibacterial microbial peptides with properties that are summarized (Ganz and Lehrer, 1999; Hancock, 1999). These (poly) peptides may be of prokaryotic or plant origin, or may be produced chemically or recombinantly (Andreu and Rivas, 1998; Ganz and Lehrer, 1999; Simmaco et al., 1998). The peptides may also belong to the class of defensins (Ganz, 1999; Ganz and Lehrer, 1999). For example, the sequences of such peptides can be found in the Antimicrobial Sequence Database at the following intemetal address:

http://www.bbcm.univ.trieste.it/~tossi/pagl.html

Such host defense peptides, or defensive peptides, are also a preferred form of the polycationic polymer of the present invention. In general, a compound allowing the activation (or downregulation) of the adaptive immune system, preferably mediated by APC (including dendritic cells), is used as the end product.

Particularly preferred for use as a polycationic agent in the present invention are antimicrobial peptides or derivatives thereof (A 1416/2000, incorporated herein by reference), particularly antimicrobial peptides derived from mammalian catelicidin, preferably human, bovine or murine catelicactin, derived from catelicidin compounds such as (human) growth hormone.

Polycationic compounds derived from natural sources include HIV-REV or HIV-TAT (derived cationic peptides, antennapedia peptides, chitosan or other chitin derivatives), or other peptides derived from these peptides or proteins by biochemical or recombinant production. Other preferred polycationic compounds are cateline or related, or derivative of cateline. For example, murine cateline is a peptide having the amino acid sequence NH ₂ -RLAGLLRKGGEKIGEKLKKIGOKIKNFFQKLVPQPECOOH. Catelin-related substances include all or parts of the catelin sequence with at least 15-20 amino acid residues. Derivatives may include substitution or modification of natural amino acids that are not among the 20 standard amino acids. In addition, other cationic residues may be introduced into such cateline molecules. It is preferred that these catelin molecules are combined with the antigen and the immunogen ODN of the present invention. Surprisingly, however, it has been shown that these catelin molecules are also effective as adjuvants for the antigen without the addition of other adjuvants. It is therefore possible to use such catelin molecules as active adjuvants in vaccine formulations with or without other immunoactivating agents.

Another preferred polycationic agent for use in the present invention is a synthetic peptide comprising at least 2 KLK motifs separated by a 3 to 7 hydrophobic amino acid linker (A 1789/2000, incorporated herein by reference).

It is very surprising that the immunostimulatory effect of the pharmaceutical composition of the present invention was significantly higher than could be expected from the sum of the effects of each individual component or even the sum of the effects of ODN or polycation with the antigen.

B and E in formula (I) are common groups for the 5 'and / or 3' ends of the nucleic acid molecules. Examples of such groups are readily available to persons skilled in the art (see, for example, "Oligonucleotides and Analogues - A Practical Approach" (1991), ed. Eckstein, Oxford University Press). For the I-ODNs of the present invention B and / or E are preferably selected independently from -H, -CH ₃ , -COCH ₃ , -OH, -CHO, phosphate, triphosphate, sulfate or thiosulfate groups, or phosphoalkyl groups, especially of length C 1 -C ₆ alkyl and / or with a terminal amino group (for example, the amino group may be used to further denote the I-ODN of the present invention, for example -PO 4 (CH ₂ ) _n -NH ₂ or -PO 4 - (CH ₂ ) _n -NH -brands). Particularly preferred as B are nucleosides, especially the 2'deoxynucleotides mentioned (i.e., without a phosphate or thiophosphate group). Alternatively, these groups may also contain linking groups to other molecules, especially carrier molecules or labels. In those forms of ODNs where ODNs are attached to solid surfaces or particles, or labels, these are surfaces, particles, labels, and the like. also part of the B and / or E groups.

Of course, any (salt) form or tautomeric form of the molecules of formula (I) are included in this formula (I).

The pharmaceutical composition of the present invention may further comprise other active ingredients, pharmaceutically active agents, especially agents that are useful in conjunction with vaccines. Preferred forms of such additional active ingredients are cytokines, anti-inflammatory agents, antimicrobials or combinations thereof.

The pharmaceutical composition of the present invention may, of course, further comprise excipients, in particular a pharmaceutically acceptable carrier, buffering agents, stabilizing agents or combinations thereof.

The relative amount of the ingredients in the present pharmaceutical composition is highly dependent on the need for an individual antigen and on the animal / human to whom the composition should be administered. Therefore, the pharmaceutical composition of the present invention preferably comprises one or more ODNs of the present invention, preferably 1 µg to 10 g, preferably 1 µg to 1 g, more preferably 1000 µg to 10 mg, especially 10 mg to 1 mg. The antigen as well as the polycationic polymer can be applied at similar dosages, preferably a range of 1 to 10,000 mg of antigen and 0.1 to 1,000 mg of polycation for vaccination.

The present compositions may be administered to a patient, for example, a vaccine candidate, in effective amounts, for example, weekly, twice weekly, or at monthly intervals. Patients to be treated with the present compositions may also be vaccinated repeatedly or only once. A preferred use of the present invention is the active immunization, especially of a human or animal without protection against a specific antigen.

The method of administration of the present composition is not critical, for example subcutaneous, intramuscular, intradermal or transdermal injection, as well as oral administration.

It is also possible to administer the present composition separately, for example by injecting the immunostimulating agent separately from the antigen / polycation preparation. Therefore, the present invention also encompasses a kit comprising a composition comprising an antigen and a polycationic polymer as one component and a composition comprising an immunostimulatory and a chemotactic agent as a second component.

The components may be applied to the same site or at the same time, but application to different sites or at different times over different time periods is also possible. It is also possible to vary the systemic and local application of the formulation or ingredients.

The details of the present invention are described by the following examples and figures, but the invention is not, of course, limited thereto.

Fig. 1 shows an immune response against ovalbumin-derived peptide OVA257.2M after injection of OVA257.264, poly-L-arginine (pR 60) and deoxyinosine I containing oligonucleotides (I-ODN) or CpG 1668. Mice were injected with hind paws , as it is stated. Four days later the draining lymph node cells ex vivo stimulated with OVA ₂₅ 7. _264th The number of IFN-g producing cells was determined 24 hours later using an ELISPOT assay. Results are expressed as spots / 10 ⁶ lymph node cells.

Fig. 2 shows the induction of systemic TNF-a after the injection of OVA _257-264, poly-L-arginine (pR 60) and deoxyinosine I-containing oligonucleotides (I-ODN) or CpG 1668. Mice were injected into the hind footpads with mixtures applied as indicated . One hour after injection, blood was collected from the tail vein and serum was prepared from the blood. Serum TNF-α concentration was determined by ELISA.

Fig. 3 shows the immune response against the OVA peptide ₂ 57_ ₂₆₄ Ovalbumin-derived after the injection of OVA _257-26 4, poly-L-arginine (pR 60) and deoxyinosine I-containing oligonucleotides (I-ODN) or CpG 1668 or GpC. Mice were injected into the hindpaws as described above. Four days later, the draining lymph node cells were ex vivo stimulated with OVA ₂ 5 ₇ . ₂₆₄ irrelevant peptide mTRP2 ₁₈₁ .i ₈₈ (2 related protein of mouse tyrosinase, VYDFFVWL) or pR 60. The number of IFN-g producing cells was determined 24 hours later using an ELISPOT assay. Results are expressed as spots / 10 ⁶ lymph node cells with a standard deviation of triplets.

Fig. 4 shows the induction of systemic TNF-a after the injection of OVA 7_ ₂₅ 6 _{4 2,} poly-L-arginine (pR 60) and deoxyinosine I-containing oligonucleotides (I-ODN) or CpG 1668. Mice were CpG in the hind paws injected with the mixture, as it is stated. One hour after injection, blood was collected from the tail vein and serum was prepared from the blood. Serum TNF-α concentration in serum was determined by cytokine-specific ELISA.

Fig. 5 shows an immune response against the OVA ₂₅₇ peptide. ₂₆₄ derived from ovalbumin after injection of TRP-2, poly-L-arginine, CpG 1668, or 20-mer sequences containing deoxyinosine. Mice were injected into the hindpaws as described above. Four days later, lymph node drainage cells were ex vivo stimulated with TRP-2, an irrelevant OVA ₂₅₇ peptide. ₂₆₄ or pR60. The number of IFN-g producing cells was determined 24 hours later using an ELISPOT assay. Results are expressed as spots / 10 ⁶ lymph node cells with a standard deviation of triplets.

Fig. 6 shows the combined injection of I-ODN and poly-L-arginine (pR 60) together with a melanoma-derived peptide.

Fig. 7 shows that combined injection of I-ODN α (pR 60) together with a melanoma-derived peptide reduces the induction of systemic TNF-α and IL-6.

Fig. 8 shows the combined injection of a random 10-mer I-ODN α (pR 60) together with a melanoma-derived peptide.

Fig. 9 shows that the combined administration of ovalbumin (OVA) with oligo-dIC26-mer ^and pR increases the production of OVA specific IgG antibodies. Mice were injected into the hindpaws as described above. Sera were collected on days 24 and 115 post-injection and assayed by ELISA for OVA specific IgG2a (A) and IgG1 (B) antibodies. Results are reported as antibody titer.

DETAILED DESCRIPTION OF THE INVENTION

Thiophosphate substituted ODNs (with phosphate substituting thiophosphate residues, hence called "thiophosphate substituted oligonucleotides") were used in all experiments because such ODNs have higher nuclease resistance (Ballas et al., 1996; Krieg et al., 1995; Parronchi et al. , 1999).

Example 1

Combined injection of various I-ODN and poly-L-arginine (pR 60) synergistically enhances the immune response against ovalbumin-derived peptide C57BI / 6 mice (Harlan / Olac)

Peptide Peptide OVA ₂₅₇ . ₂₆₄ (SIINFEKL), a MHC Class I (H-2Kb) restricted epitope of chicken ovalbumin (Rotzschke et al., 1991), was synthesized using standard F-moc chemical solid-phase synthesis, purified by HPLC, and analyzed by mass spectroscopy for purity. Dose: 300 mg / mouse

Poly-L-arginine 60 CpG-ODN 1668

I-ODN 1

I-ODN 2

I-ODN 3 (pR60) Poly-L-arginine with an average degree of polymerization of 60 arginine residues; Sigma. A dose of 100 mg / mouse thiophosphate substituted ODN containing the CpG motif: tcc atg acg ttc ctg atg ct was synthesized by NAPS GmbH, Gottingen. A dose of 5 nmol / mouse thiophosphate substituted ODN containing deoxyinosine: tcc ati tci ttc ctg atg ct was synthesized by NAPS GmbH, Gottingen. A dose of 5 nmol / mouse thiophosphate substituted ODN containing deoxyinosine: tcc atg and ttc ctg atg ct was synthesized by NAPS GmbH, Gottingen. A dose of 5 nmol / mouse thiophosphate-substituted ODN containing deoxyinosine: tcc ati tci ttc ti ati ct was synthesized by NAPS GmbH, Gottingen. Dose 5 nmol / mouse

Experimental groups (5 mice per group)

1. OVA257-264

2. OVA257-264 + pR 60

3. OVA ₂₅ 7_264 + CpG 1668

4. OVA ₂ 5 ₇ _264 + I - ODN 1

5. OVA ₂ 57-264 + I - ODN 2

6. OVA ₂ 57-264 + I - ODN 3

7. OVA ₂ 57_ ₂ 64 + CpG 1668 + pR 60

8. OVA ₂ 57_ ₂ 64 + I-ODN 1 + pR 60

9. OVA ₂₅₇ -264 + I-ODN 2 + pR 60

10. OVA 57_ _{2 2} 64 + I-ODN 3 + pR 60

On day 0, mice were injected into each hindpaw with a total volume of 100 ml (50 ml per paw) containing the compounds. Animals were sacrificed 4 days after injection and popliteal lymph nodes were collected. The lymph nodes were passed through a 70 mm cell sieve and washed twice with DMEM medium (Gibco BRL) containing 5% fetal calf serum (FCS, Sigma). Cells were adjusted to 3 x 10 ⁶ cells / ml in DMEM / 5% / FCS. ELISPOT IFNg analysis was performed in a triplet as described (Miyahira et al., 1995). This method is a widely used procedure for quantifying antigen-specific T cells. Lymphocytes were stimulated ex vivo with control medium, peptide OVA ₂₅₇ . ₂₆ 4 or Concavalin A (Con A). Spots representing individual IFN-γ producing T cells were counted and the number of background spots was subtracted from all samples. The high number of spots detected after stimulation with Con A (data not shown) indicate the good state of the lymphocytes used. For each experimental group of mice, spot numbers / 10 ⁶ cells are illustrated in Figure 1.

One hour after injection, blood was collected from the tail vein and serum was prepared to determine the induction of systemic TNF-α using an ELISA assay (Figure 2).

Example 2

The exchange of guanosine by desoxy-inosine converts the non-immunogenic GpC sequence to a highly immunogenic, particularly in combination with poly-L-arginine (pR60).

mice

peptide

Poly-L-Arginine 60 CpG-ODN 1668 CpG-ODN

I-ODN

I-ODN 10

Harlan / Olac C57BI / 6

OVA257-264 (SIINFEKL), MHC class 1 (H-2Kb) restricted chicken ovalbumin epitope (Rotzschke et al., 1991) was synthesized using standard F-moc solid-phase chemical synthesis, purified by HPLC and analyzed by mass spectrometry. spectroscopy for purity. Dose: 300 µg / mouse (pR60) Poly-L-arginine with an average degree of polymerization of 60 arginine residues; Sigma. A dose of 100 µg / mouse thiophosphate substituted ODN containing the CpG motif: tcc atg and ttc ctg atg ct were synthesized by NAPS GmbH, Gottingen. A dose of 5 nmol / mouse thiophosphate substituted ODN containing a non-immunogenic GpC motif: tcc atg agc ttc ctg atg ct was synthesized by NAPS GmbH, Gottingen. A dose of 5 nmol / mouse thiophosphate substituted ODN containing deoxyinosine: tcc atg and ttc ctg atg ct was synthesized by NAPS GmbH, Gottingen. A dose of 5 nmol / mouse thiophosphate-substituted ODN containing deoxyinosine: tcc ati and ttc atti-ct was synthesized by NAPS GmbH, Gottingen. Dose 5 nmol / mouse

Experimental groups (5 mice per group)

OVA ₂ 57_ ₂ 64

57_ _{2 2} OVA + pR 64 60

OVA ₂ 57_ ₂₆₄ + CpG 1668

OVA ₂₅ 7_ ₂₆ 4 + GpC

OVA ₂₅₇ -264 + I-ODN 9

OVA ₂₅₇ -264 + I-ODN 10

OVA ₂ 57. ₂₆₄ + CpG 1668 + pR60

OVA ₂ 57_ ₂₆₄ + GpC + pR 60

OVA ₂₅₇ . ₂₆₄ + I-ODN 9 + RP 60

OVA ₂₅₇ . ₂₆ 4 + I-ODN 10 + p 60

On day 0, mice were injected into each hindpaw with a total volume of 100 µl (50 µl per paw) containing the compounds. Animals were sacrificed 4 days after injection and popliteal lymph nodes were collected. The lymph nodes were passed through a 70 µm cell sieve and washed twice with DMEM medium (Gibco BRL) containing 5% fetal calf serum (FCS, Sigma). Cells were adjusted to 3 x 10 ⁶ cells / ml in DMEM / 5% / FCS. ELISPOT IFNg analysis was performed in a triplet as described (Miyahira et al., 1995). This method is a widely used procedure for quantifying antigen-specific T cells. Lymphocytes were stimulated ex vivo with medium serving as a control peptide Ova ₂ 57_ ₂₆₄ / irrelevant peptide MTRP-2 ₈ 8 _g i_i (protein-2 mouse tyrosinase related, VYDFFVWL), pR 60 and Concanavalin A (Con A). Spots representing individual IFN-γ producing T cells were counted and the number of background spots was subtracted from all samples. The high number of spots detected after stimulation with Con A (data not shown) indicate the good state of the lymphocytes used. For each experimental group of mice, spot numbers / 10 ⁶ cells are illustrated in Figure 3, standard deviations of ex vivo stimulated triplets are shown. One hour after injection, blood was collected from the tail vein and serum was prepared to determine the induction of systemic TNF-α and IL-6 using an ELISA assay (Figure 4).

Example 3

The combined injection of random 20-mer sequences containing desoxyinosine and a melanoma-derived peptide induces a strong immune response against the peptide, which can be further enhanced by co-administration of poly-L-arginine (pR 60).

C57BI / 6 Mice (Harlan / Olac)

Peptide The TRP-2 peptide (VYDFFVWL), MHC class 1 (H-2K ^b ) restricted epitope of the related murine tyrosinase-related protein-2 (Bllom et al., 1997), was synthesized using standard F-moc solid-phase chemical synthesis, purified by HPLC and analyzed by mass spectroscopy for purity. Dose: 300 µg / mouse

Poly-L-arginine 60 (pR60) Poly-L-arginine with an average degree of polymerization of 60 arginine residues; Sigma. Dose 100 µg / mouse

CpG-ODN 1668 thiophosphate substituted ODN containing a CpG motif: tcc atg and ttc ctg atg ct were synthesized by NAPS GmbH, Gottingen. A dose of 5 nmol / mouse wdi thiophosphate substituted ODN: nhh hhh wdi nhh hhh hhh wn was synthesized by NAPS GmbH, Gottingen. A dose of 5 nmol / mouse wdidine thiophosphate-substituted ODN: nhh hhh wdi nhh hhh hhh wn was synthesized by NAPS GmbH, Gottingen. A dose of 5 nmol / mouse wdid thiophosphate substituted ODN: nhh hhh wdi dhh hhh hhh wn was synthesized by NAPS GmbH, Gottingen. The 5 nmol / mouse thiophosphate substituted ODN dose: nhh wdi did hhh hdi ddi dh were synthesized by NAPS GmbH, Gottingen. Dose 5 nmol / mouse

Experimental groups (5 mice per group)

TRP-2

2. TRP-2 + pR60

3. TRP-2 + CpG1668

4. TRP-2 + wdi

5. TRP-2 + wdidine

6. TRP-2 + wdid

7. TRP-2 + wdidide

8. TRP-2 + CpG1668 + pR60

9. TRP-2 + wdi + pR60

10. TRP-2 + widin-pR + 60

11. TRP-2 + wdid + pR60

12. TRP-2 + widide + pR60

On day 0, mice were injected into each hindpaw with a total volume of 100 µl (50 µl per paw) containing the compounds. Animals were sacrificed 4 days after injection and popliteal lymph nodes were collected. The lymph nodes were passed through a 70 µm cell sieve and washed twice with DMEM medium (Gibco BRL) containing 5% fetal calf serum (FCS, from Sigma). Cells were adjusted to 3X

10 ⁶ cells / ml in DMEM / 5% / FCS. ELISPOT IFN-γ analysis was performed in a triplet as described (Miyahira et al., 1995). This method is a widely used procedure for quantifying antigen-specific T cells. Lymphocytes were stimulated ex vivo with medium serving as a control peptide TRP-2, an irrelevant peptide OVA ₂ 5 _{7th 264} , pR 60 and Concavalin A (Con A). Spots representing individual IFN-γ producing T cells were counted and the number of background spots was subtracted from all samples. The high number of spots detected after stimulation with Con A (data not shown) indicate the good state of the lymphocytes used. For each experimental group of mice, spot numbers / 10 ⁶ cells are illustrated in Figure 5, standard deviations of ex vivo stimulated triplets are shown.

Example 4

The combined injection of I-ODN and poly-L-arginine (pR 60) synergistically enhances the immune response against the melanoma-derived peptide.

Experimental groups (5 mice per group)

1. TRP-2181-188

2. TRP-2.81.188 + PR 60

3. TRP-2.81.188 ⁺ CpG 1668

4. TRP-2, s, s, s + I-ODN 2

5. TRP-2.81-188 + CpG 1668 + pR 60 6. TRP-2 18M88 + I-ODN 2 + pR 60

On day 0, mice were injected into each hindpaw with a total volume of 100 µl (50 µl per paw) containing the compounds. Animals were sacrificed 4 days after injection and popliteal lymph nodes were collected. The lymph nodes were passed through a 70 µm cell sieve and washed twice with DMEM medium (Gibco BRL) containing 5% fetal calf serum (FCS, Sigma). Cells were adjusted to 3 x 10 ⁶ cells / ml in DMEM / 5% / FCS. ELISPOT IFN-γ analysis was performed in a triplet as described (Miyahira et al., 1995). This method is a widely used procedure for quantifying antigen-specific T cells. Lymphocytes were stimulated ex vivo with medium serving as a control peptide TRP 2.81 to 188, ₂₅ irrelevant peptide _OVA-26 7. 4 and Concanavalin A (Con A). Spots representing individual IFN-γ producing T cells were counted and background spots were subtracted from all samples. The high number of spots detected after stimulation with Con A (data not shown) indicate the good state of the lymphocytes used. For each experimental group of mice, spot numbers / 10 ⁶ cells are illustrated in Figure 6, standard deviations of ex vivo stimulated triplets are shown.

One hour after injection, blood was collected from the tail vein and serum was prepared to determine the induction of systemic TNF-α and IL-6 using specific ELISA assays (Figure 7).

Example 5

The combined injection of random 10-mer I-ODN and poly-L-arginine (pR 60) synergistically enhances the immune response against the melanoma-derived peptide.

Experimental groups (5 mice per group). TRP-2.81-188. TRP-2181-188 + pR60

3. TRP-2.81-188 + CpG 1668

4. TRP-2, s, -188 + ODN 17

5. TRP-2.81-188 + CpG 1668 + pR60

6. TRP-2.81-188 + ODN 17 + pR60

On day 0, mice were injected into each hindpaw with a total volume of 100 µl (50 µl per paw) containing the compounds. Animals were sacrificed 4 days after injection and popliteal lymph nodes were collected. The lymph nodes were passed through a 70 µm cell sieve and washed twice with DMEM medium (Gibco BRL) containing 5% fetal calf serum (FCS, Sigma). Cells were adjusted to 3 x 10 ⁶ cells / ml in DMEM / 5% / FCS. ELISPOT IFN-γ analysis was performed in a triplet as described (Miyahira et al., 1995). This method is a widely used procedure for quantifying antigen-specific T cells. Lymphocytes were stimulated ex vivo with medium serving as a control peptide TRP 2,8i.i88> irrelevant peptide OVA ₂ 57- ₂ 64 and Concanavalin A (Con A). Spots representing individual IFN-γ producing T cells were counted and background spots were subtracted from all samples. The high number of spots detected after stimulation with Con A (data not shown) indicate the good state of the lymphocytes used. For each experimental group of mice, spot numbers / 10 ⁶ cells are illustrated in Figure 8, standard deviations of ex vivo stimulated triplets are shown.

C57BI / 6 Mice (Harlan / Olac)

Peptide The TRP-2 peptide (VYDFFVWL), an MHC class 1 (H-2Kb) restricted epitope of the protein-2 related mouse tyrosinase (Bllom et al., 1997), was synthesized using standard 11.

Poly-L-arginine CpG-ODN 1668

ODN 17

mice

peptide

Poly-L-arginine 60 CpG-ODN 1668

I-ODN 2 of its solid-phase F-moc chemical synthesis, purified by HPLC and analyzed by mass spectroscopy for purity. Dose: 100 µg / mouse (pR60) Poly-L-arginine with an average degree of polymerization of 60 arginine residues; Sigma. A dose of 100 µg / mouse thiophosphate substituted ODN containing the CpG motif: tcc atg and ttc ctg atg ct were synthesized by NAPS GmbH, Gottingen. A dose of 5 nmol / mouse thiophosphate substituted ODN containing deoxyinosine: hhh wdi dhh h was synthesized by NAPS GmbH, Gottingen, (h = CAT, w = AT, d = GAT). Dose: 10 nmol / mouse

Harlan / Olac C57BI / 6

Peptide TRP-2 (VYDFFVWL), MHC class 1 (H-2K ^b ) restricted epitope of protein-2 related mouse tyrosinase (Bllom et al., 1997) was synthesized using standard F-moc solid-phase chemical synthesis, purified by HPLC and analyzed by mass spectroscopy for purity. Dose: 100 µg / mouse (pR60) Poly-L-arginine with an average degree of polymerization of 60 arginine residues; Sigma. A dose of 100 µg / mouse thiophosphate substituted ODN containing the CpG motif: tcc atg and ttc ctg atg ct were synthesized by NAPS GmbH, Gottingen. A dose of 5 nmol / mouse thiophosphate substituted ODN containing deoxyinosine: tcc atg and ttc ctg atg ct was synthesized by NAPS GmbH, Gottingen. Dose: 5 nmol / mouse

Example 6

The combined administration of oligo-deoxyIC ₂ 6. _mer and poly-L-arginine (pR) enhances the ovalbumin (OVA) specific humoral response.

C57BI / 6 Mice (Harlan / Olac)

Ovalbumin (OVA) Chicken Egg Ovalbumin, Grade V, SIGMA Chemicals, A-5503, Lot 54H7070. Dose 50 µg / mouse

Poly-L-arginine 60 (pR60) Poly-L-arginine with an average degree of polymerization of 60 arginine residues; Sigma Chemicals, P-4663, lot 68H5903. The dose of 100 / mouse Oligo-deoxy IC, 26-mer (oligo-dIC _{26th RAER)} oligo-dIC _{26th mer} was synthesized by standard phosphoamidide chemistry on a 4 µm scale and purified by HPLC (NAPS, Germany).

Experimental groups (4 mice per group)

1. OVA + 0lg0-dIC ₂ 6-mer + PR

2. OVA + 0lÍg0-dic _two 6-mer

3. OVA + pR

4. OVA

On day 0, mice were injected into each hindpaw with a total volume of 100 µl (50 µl per paw) containing the compounds. 24 days after injection, serum was collected and examined by ELISA for the presence of OVA specific antibodies. These results show that injection of OVA in combination with oligo-dlC and pR increases the production of OVA specific IgG antibodies compared to injection of OVA with each substance alone (Figure 13A, B). Interestingly, both IgG2a and IgG1 titers were increased after a single injection of OVA with oligo-dIC / pR, indicating both Th1 and Th2 cells. However, only elevated IgG2 levels in the serum of mice after OVA and oligo-dIC / pR injection were still detectable after 115 days.

These data demonstrate that the combined injection of OVA with oligo-dlC and pR enhances the OVA specific humoral response. This response is characterized by the production of isotypes of antibodies induced by both Th1 and Th2 cells in the early phase, but later mainly by antibodies induced by Th1 cells.

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Claims (10)

PATENT CLAIMS Use of an oligodeoxynucleic acid (ODN) molecule having the structure defined by formula (I) NMP _b -E (X) wherein any X is O or S; any of the NMP residues is 2'-deoxynucleoside monophosphate or -monothiophosphate selected from deoxyadenosine-, deoxyguanosine-, deoxyinosine-, deoxycytosine-, deoxyuridine-, deoxythymidine-, 2-methyldeoxyinosine-, 5-methyl-deoxycosine- deoxycosine, -, 2-aminodeoxyribosapurine, 6-S-deoxyguanine, 2-dimethyldeoxyguanosine or N-isopentenyldeoxyadenosine monophosphate or monothiophosphate, NUC is a 2'-deoxynucleoside selected from deoxyadenosine-, deoxyguanosine-, deoxyinosine-, deoxycytosine-, deoxyuridine-, deoxythymidine-, 2-methyldeoxyinosine-, 5-methyldeoxycytosine-, deoxypseudouridine-, deoxypyuribine-, deoxyribosine-, deoxyrurosine, 6-deoxyrolidine, -deoxyguanine-, 2-dimethyldeoxyguanosine- or N-isopentenyldeoxyadenosine, and b are integers from 0 to 100, with the proviso that a + b is a number between 4 and 150, B and E are common groups for the 5 'or 3' ends of the nucleic acid molecules, for the manufacture of an immunostimulatory pharmaceutical composition. Use according to claim 1, wherein any NMP is selected from deoxyadenosine, deoxyguanosine, deoxyinosine, deoxycytosine, deoxyuridine, deoxythymidine, 2-methyldeoxyinosine, 5-methyl-deoxycytosine monophosphate or monothiophosphate. Use according to claim 1 or 2, wherein a + b is between 10 and 60, preferably between 15 and 40. The use according to any one of claims 1 to 3, wherein at least one of X 1 and X ₂ is S and at least one of X ₃ and X ₄ is O and preferably any of the NMP is nucleoside monothiophosphate. 5, further comprising further active ingredients, in particular cytokines, anti-inflammatory agents, antimicrobials or combinations thereof. Pharmaceutical preparation according to any one of claims 11 to 14, characterized in that it further comprises excipients, in particular a pharmaceutically acceptable carrier, buffering agents, stabilizing agents or combinations thereof. Use according to any one of claims 1 to 4, wherein the ODN comprises the sequence hhh wdi dhh h, nhh hhh wdi nhh hhh hhh wn, nhh wdi din hhh hdi ndi nh, nhh hhh wdi dhh hhh hhh wn or nhh wdi did hhh hdi ddi dh, wherein any n is 2'-deoxynucleoside monophosphate or -monothiophosphate selected from deoxyadenosine, deoxyguanosine-deoxyinosine, deoxycytosine- or deoxythymidine monophosphate, or -monothiophosphate, any of which is 2'-deoxynucleoside monophosphate, or deoxycytosine- or deoxythymidine monophosphate, or -monothiophosphate, i is deoxyinosine monophosphate or -monothiophosphate, any of which is 2'-deoxynucleoside monophosphate or a monothiophosphate selected from deoxyadenosine-, or deoxythymidine monophosphate, or -monothiophosphate, and any of d is 2'-deoxynucleoside monophosphate or -monothiophosphate selected from deoxyadenosine, deoxyguanosine or deoxythymidine monophosphate, or -monothiophosphate. Use according to any one of claims 1 to 5, wherein said ODN comprises at least one 2'-deoxycytosine monophosphate or -monothiophosphate, 3'-adjacent to 2'-deoxyinosine monophosphate or -monothiophosphate, in particular deoxyinosine deoxycytosine 26-mer. Use according to any one of claims 1 to 7, wherein said ODN comprises the sequence wdi, wdid, wdidin or wdidid, wherein w, d, i and n are defined. Use according to any one of claims 1 to 6, wherein said ODN comprises a sequence of gacitt, in particular tcc atg and ttc ctg atg ct, iacitt, gaictt, iaictt, wherein a is deoxyadenosine monophosphate or monothiophosphate, g is deoxyguanosine monophosphate or - monothiophosphate, i is deoxyinosine monophosphate or -monothiophosphate, c is deoxycytosine monophosphate or -monothiophosphate and t is deoxythymidine monophosphate or -monothiophosphate. Use according to any one of claims 1 to 8, wherein B and E are independently selected from -H, -CH ₃ , -COH, -COCH ₃ , -OH, -CHO, -PO 4, -PSO ₃ , -PS ₂ O _2, -PS ₃ O, -PS _4, -SO _3, -PO4 _(CH2) ^ - NH ₂ or -PO ₄ (CH ₂₎ ,. ₆ -NHtag. Use according to any one of claims 1 to 9, wherein said immunostimulatory pharmaceutical composition is a vaccine. Pharmaceutical preparation of any one of claims 1 to 9. Pharmaceutical preparation of any one of claims 1 to 9. Pharmaceutical preparation of any one of claims 1 to 9 and an antigen. Pharmaceutical preparation according to claim 11 or 12, characterized in that it further comprises a polycationic polymer, preferably a polycationic peptide, in particular polyarginine, polylysine or anti- characterized by mark the task by characterized by a microbial peptide, especially an antimicrobial peptide derived from mammalian cathelicidin, a synthetic peptide comprising 2 KLK-motifs separated by a linker of 3 to 7 hydrophobic amino acids, or growth hormone, especially human growth hormone. Pharmaceutical preparation according to any one of claims 11 to 13, characterized by Pharmaceutical preparation according to any one of claims 11 to 15, characterized in that it contains 1 ng to 1 g, preferably 100 ng to 10 mg, in particular 10 mg to 1 mg of one or more ODNs according to any one of claims 1 to 9.